PHOTO II

Photo II was one of the experiments that composed the scientific payload of the satellites Foton M2 and M3 launched from the Cosmodrome of Baikonour (Kazakhstan), in 2005 and 2007 (see figure 2, showing the Soyuz before and after the flight). The Photo project investigated the possibility of using oxygenic photosynthetic microorganisms on long- term Space flights as a source of food, oxygen and nutraceutical compounds. The goal pursued was to assess the effects of the Space environment on various mutated microorganisms in order to select resistant-tolerant strains and determine the production of compounds with anti-oxidant properties resistant to Space ionizing radiation.

Microgravity and ionizing radiation, which can influence the viability and performance of these organisms, are the critical points to resolve in utilizing plants or algae-based life supporting systems.

During the missions, Photo II monitored automatically the photosynthetic activity of several Chlamydomonas reinhardtii strains, unicellular green algae, carrying mutations in the D1 protein of Photosystem II [9, 10].

The D1 protein is a subunit involved in the formation of the core complex of PSII and it has a fundamental role in the photosynthetic process.

Space ionizing radiation, various stress conditions and the absence of gravity can damage the D1 protein. Testing the radio resistance of D1-protein- mutants allows the amino acidic substitutions that are able to improve the tolerance of the microorganisms to the Space environment to be identified [9]. Microalgae are particularly suited as a regenerative-life supporting system as they have a low sensitivity to microgravity, a short life cycle, are easy to cultivate in photobioreactors and have high biomass productivity. They are also a rich source of secondary metabolites with anti-oxidant properties to provide a nutraceutical anti-oxidant-enriched biomass as a dietary supplement for the crew of spacecrafts. Some organic pigments play a key role in protecting photosynthesis under stress conditions; particularly xanthophylls that are oxygen-containing carotenoids, i.e. zeaxanthin, antheraxanthin, violaxanthin, involved in the photo protection of the photosynthetic apparatus. When the photosynthetic organisms are illuminated by a strong light, which exceeds their capacity for photosynthesis, the excess energy can be harmful for the photosynthetic apparatus. Under these conditions, the non-dissipating pigment violaxanthin is rapidly converted, via the intermediate antheraxanthin, to zeaxanthin, that has high photoprotective properties, dissipating the energy in excess [11]. This conversion cycle could be the strategy adopted by the photosynthetic organisms to survive in Space [unpublished results].

Photo II, a multicell fluorescence biosensor. In recent years, chlorophyll a (Chl a) fluorescence has become so essential in physiological and ecophysiological studies, that all investigations concerning photosynthetic performance of algae and plants are considered complete only if accompanied by fluorescence data. The great success of this technique is attributable to the fact that it gives the possibility of determining the physiological state of photosynthetic organisms, under conditions in which other methods would fail and, above all, in an instantaneous and non- intrusive manner. It means that performing fluorescence analysis, unlike most analytical techniques, does not always require sample preparation steps and therefore direct measurements can be often performed [12].

The basics of chlorophyll fluorescence have been extensively discussed over the last decades.

Substantially, when photosynthetic organisms absorb light, a chain of reactions, overall known as photosynthesis, begins. The process starts with the absorption of photons of light by Chl molecules surrounding the two photosystems (PSII and PSI) organized in light harvesting complexes in the photosynthetic apparatus [13]. This creates resonance energy that is transferred to the neighbouring Chl molecules, reaching finally the reaction centre (RC) embedded in the core complex [14]. Consequently, another series of reactions involving different mobile carriers occur, leading to the production of energy rich molecules and reducing equivalents, which are needed to convert carbon dioxide to carbohydrate via the Calvin cycle.

In this way, by several physical and chemical mechanisms, radiation energy is transformed into chemical energy. However, not all radiation energy follows this fate, the excess of it being dissipated as heat (around 18%) or re-emitted as fluorescence (1- 2% of the total light absorbed). The three processes (photosynthesis, heat production and Chl fluorescence) occur in competition, in the way that any increase in the efficiency of one will produce a decrease in the yield of the other two [12, 13, 15, 16]. Thus, if photochemistry is blocked (for instance, due to the presence of ionizing radiation), the yield of non-photochemical reactions proportionally rises, giving indirect information about the overall photosynthetic performance. Since at room temperature the major contribution to Chl a fluorescence comes from PSII, whereas that of PSI is smaller (around 10-25% of the Initial fluorescence F0 ), measuring Chl a fluorescence also represents a valid tool for indirect investigation on the state of PSII.

In experiment Photo II measures were based on chlorophyll fluorescence induction, also known as fluorescence transient or Kautsky‟s effect [12, 13, 15].

As shown in figure 3, Photo II is composed of four identical, independent units, each of them powered by two batteries in series (7.5 V). Every unit is composed of two separated modules, each one made up of three optical cells where the fluorescence measurements are carried out.

In each cell, the measurement system is composed of four red light LEDs and an optical fluorescence sensor that provides hourly measurements. The

exciting light pulse from the red LEDs is for 6 seconds with an intensity peak at λ=660 nm, inducing the chlorophyll fluorescence; the average intensity of the exciting red lights is ~800 µmol m^-2 s^-1, as measured at the centre of the cells using a quantum radiometer. Before starting each measurement session, the samples are dark adapted for 15 minutes to allow a complete reduction of Photosystem II. A filter is mounted on the top of the optical detector that allows the high- pass transmission of the fluorescence light with wavelengths λ>690 nm. The fluorescence measurements are then digitized and recorded in a non-volatile memory (NVM), where more than 500 measurements can be stored.

Fig. 2. The above to below: Capsule Foton before flight; with Photo II (while arrow) inside the capsule;

Foton after landing.

In each measurement cell the living conditions are provided by two white light LEDs that are switched on continuously for 7 h out of a 24 h period (17

hours of dark) and guarantee the photoperiod necessary for the survival of the samples.

Fig. 3. The Photo II fluorimeter used in Foton Space missions; the main components are shown and highlighted. External box was built by Kayser-Italia, while electronics were built by Italian companies:

Biosensor, Carso.

The intensity of the white lights can be set up to a maximum of 250 µmolm^-2s^-1 as measured at centre of each cell.

The material used for the containers of the biological samples is Delrin and the transparent window exposed to the light is made of

Polycarbonate and a steel frame is used to seal the cell. Black Delrin is compatible with the biological material and guarantees optical isolation among the cells. A gasket of silicone provides perfect sealing, thus avoiding any contamination of the biological material after being placed in the container.

Finally a set of eight independent thermometers to measure the temperature inside the device complete the electronics of Photo II.

2.1 Photo II Electrical and electronic system Space is a very hostile environment for electronic components. The presence of radiation of different types, intensity and energy can cause various effects some of which are potentially dangerous for the mission [17, 18].

The radiation effects due to long-term exposure in the space are known as total dose effects. This accumulates energy (in the form of ionized atoms) over time in the target material leading to an increase of leaking currents in isolators and transistors, charge trapping in MOS gate oxide (potentially leading to transistor failure) and loss of internal chip isolation.

Another effect of radiation is caused when a single high-energy particle hits an electronic device. It leaves behind a column of ionized material that is like a conducting wire suddenly inserted into the device disturbing the currents and electrical fields inside it. Therefore, electronic systems for Space applications must be carefully designed. Shielding is crucial but it is not enough to resolve just this issue; devices, known as radiation hardened or called “Rad-Hard”, must be used to withstand conditions in Space [18].

NASA and ESA have programs for testing and cataloguing components for Space missions. These are the NASA Electronics Parts and Packaging Program (http://nepp.nasa.gov/index.cfm) and the European Space Components Coordination (https://spacecomponents.org/). Lists of approved components are published periodically (GFSC Preferred Part list in NASA and ESC Qualified Parts List in ESA) and these were used as the basis for the development of Photo II.

The electronic system is composed of three specific function cards. The Photo II control board is based on a 32-bits x486 AMD processor (Elan SC410).

This processor is a low power CPU and works at a

frequency of 33MHz. Externally a 2MB flash program memory and a 8MB RAM memory have been provided.